tbuckley/cabot-search huggingface dataset:
Abstract 3529: <i>EWSR1-WT1</i> isoform selectivity, DNA binding, and druggable targets: unpacking the biology of desmoplastic small round cell tumor
Authors: Justin W. Magrath, Ilon A. Goldberg, Alifiani B. Hartono, Sean B. Lee
Journal: Cancer Research (2023)

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare pediatric cancer caused by the EWSR1-WT1 fusion gene. No targeted therapies have been established and multimodal therapy is ineffective with a 5-year survival rate of 15-25%. Many questions about the biology of DSRCT remain unanswered including the critical genes regulated by the EWSR1-WT1 fusion gene, the role of the - and +KTS fusion gene isoforms produced by alternative splicing, and the relationship between EWSR1-WT1 binding and gene expression alterations. To address these questions, we established four doxycycline-inducible DSRCT cell lines that selectively knockdown the expression of EWSR1-WT1. RNA-seq analysis on these cell lines with or without EWSR1-WT1 knockdown identified commonly altered genes and pathways. To illuminate the role of the EWSR1-WT1 -KTS and +KTS isoforms, the -KTS, +KTS, or both isoforms were overexpressed in a mesothelial cell line, a hypothesized cell of origin for DSRCT. Remarkably, different gene expression alterations were observed between the -KTS and +KTS isoforms. While overexpression of the -KTS isoform upregulated 765 genes and downregulated 366 genes, overexpression of the +KTS isoform upregulated 203 genes and downregulated 25 genes. Gene set enrichment analysis showed EWSR1-WT1 regulated gene signatures are recapitulated by overexpression of the -KTS isoform or both +/-KTS isoforms, but not the +KTS isoform alone, suggesting the -KTS isoform predominantly regulates gene expression in DSRCT. To interrogate the influence of direct and indirect gene regulation by EWSR1-WT1, ChIP-seq and CUT&amp;RUN data were integrated into our RNA-seq analysis. Across each of our four DSRCT cell lines, approximately 25% of EWSR1-WT1 bound peaks led to upregulation, 10% led to downregulation, and 65% led to no gene expression alteration. EWSR1-WT1 binding accounted for 33% of EWSR1-WT1 commonly upregulated genes but only 4.2% of commonly downregulated genes. Upregulated EWSR1-WT1 bound genes were more likely to be bound in the promoter or first intron and coincide with Znf263 or Hoxb4 motifs. To evaluate the functional role of EWSR1-WT1 targets in DSRCT biology, a screen of the 24 druggable EWSR1-WT1 targets was conducted and a number of novel and known target genes were identified as important for DSRCT survival. Current work is verifying these targets with gene knockdown and evaluating their function in vivo. Citation Format: Justin W. Magrath, Ilon A. Goldberg, Alifiani B. Hartono, Sean B. Lee. EWSR1-WT1 isoform selectivity, DNA binding, and druggable targets: unpacking the biology of desmoplastic small round cell tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3529.
Abstract 6145: Alternative splicing of GSDMB modulates killer lymphocyte-triggered pyroptosis
Authors: Qing Kong, Shiyu Xia, Xingxin Pan, Kaixiong Ye, Zhouyihan Li, Haoyan Li, Nidhi Sahni, S. Stephen Yi, Xing Liu, Hao Wu, Judy Lieberman, Zhibin Zhang
Journal: Cancer Research (2023)

Abstract Killer lymphocyte-mediated tumor cell killing is a vital and final step of anti-tumor protective immunity. Killer cells, including cytotoxic CD8 T cells and Natural Killer (NK) cells, deliver granzymes (Gzm) into target cells, which normally activates noninflammatory cell death, but becomes inflammatory when target cells express the gasdermin (GSDM) proteins responsible for pyroptosis. GzmA and GzmB cleave and activate GSDMB and GSDME, respectively. However, the role of GSDMB in anti-tumor immunity is unclear - GSDMB pore formation is controversial and GSDMB expression has been linked to both anti- and pro-tumor functions. Here we found that GSDMB splicing variants are functionally distinct. Cleaved N-terminal (NT) fragments of isoforms 3 and 4 caused pyroptosis, but isoforms 1, 2 and 5, which deleted or modified exon 6, did not. Structural analysis based on AlphaFold predictions and the cryoEM structures of GSDMA3 and GSDMD showed that pore formation required the formation of a functional stabilizing belt structural element near the C-terminus of the cleaved N-terminal fragment, which was disrupted in the non-cytotoxic alternatively spliced isoforms. NK attack of GSDMB3-expressing cells caused pyroptosis, but GSDMB4 cells died mostly by apoptosis, and GSDMB1/2 cells died only by apoptosis. GSDMB4 partially resisted NK cell-triggered cleavage, suggesting that only GSDMB3 is fully functional. GSDMB1-3 were the most abundant isoforms in tested cells and were similarly induced by IFNγ and a chemotherapy drug methotrexate. Expression of cytotoxic GSDMB3/4, but not GSDMB1/2, was associated with better outcome in bladder and cervical cancer, suggesting GSDMB-mediated pyroptosis was protective in those tumors. Our study uncovers a novel role of alternative splicing in modulating caner-associated pyroptosis, and suggests that tumors may manipulate GSDMB splicing to avoid pyroptosis. Thus, modulating GSDMB splicing to increase cytotoxic GSDMB variants but suppress non-cytotoxic GSDMB isoforms could improve anti-tumor immunity and have therapeutic utility. Citation Format: Qing Kong, Shiyu Xia, Xingxin Pan, Kaixiong Ye, Zhouyihan Li, Haoyan Li, Nidhi Sahni, S. Stephen Yi, Xing Liu, Hao Wu, Judy Lieberman, Zhibin Zhang. Alternative splicing of GSDMB modulates killer lymphocyte-triggered pyroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6145.
Abstract 615: Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways
Authors: Katrina van Raay, Michelle Kriner, Jason Reeves, Erin Piazza, Hargita Kaplan, John Vivian, Francis Fernandez, Margaret Hoang, Joseph Beechem
Journal: Cancer Research (2023)

Abstract Spatial distribution of T cells is key in understanding the escape of tumors from immune surveillance via the adaptive immune response, including interactions between immune cells and the surrounding tumor microenvironment. T cells are critical to the adaptive immune response to pathogens and cancers, mediating an antigen-specific response through both specificity and diversity of T cell receptor (TCR) clonotypes. Many methods exist to determine specific clonotypes and overall TCR diversity present from bulk tissues or sorted cell populations; however, nearly all fail to capture spatial orientation and arrangement of T cells engaging with their microenvironment, and most require large amounts of starting material from precious samples. Here, we present a TCR expression profiling panel for the GeoMx® Digital Spatial Profiler that can be combined with the GeoMx Cancer Transcriptome Atlas (CTA) or Human Whole Transcriptome Atlas (WTA) on archival formalin-fixed paraffin embedded (FFPE) tissue specimens. This represents the first commercial spatial expression profiling assay for the simultaneous quantification of TCR constant, variable, and joining segments in situ. We show reliable sensitivity and specificity (&amp;gt;90%) with respect to orthogonal sequencing and robust detection of TCR chains with evidence of clonal expansion and CD8 infiltration across tumor regions in colorectal cancer tissue. These events also corresponded to increased signatures of exhaustion from the T cells and suggest that the T cells resident in or near the tumor are tumor-specific and poised for activation via checkpoint blockade. Signaling pathways and tumor-specific signatures were also evaluated to look for mechanisms through which tumor cells respond to T cell infiltration. We further validated the performance of the TCR probe pool in cell pellet arrays with orthogonal TCR sequencing, tonsil and colorectal cancer tissues. Together, the combination of our TCR add-on panel with the CTA or WTA illuminates T cell phenotypes, signaling pathways, population dynamics, and transcriptomic changes, yielding an unparalleled view of the T cell response in any context. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Katrina van Raay, Michelle Kriner, Jason Reeves, Erin Piazza, Hargita Kaplan, John Vivian, Francis Fernandez, Margaret Hoang, Joseph Beechem. Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 615.
Abstract 4688: Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay
Authors: Erinn A. Parnell, Jiong Fei, Harry Nunns, Eric Leones, Elaine Yeung, Blair Russell, Flora Sahafi, Qingyan Au
Journal: Cancer Research (2023)

Abstract As a promising alternative platform for cellular immunotherapy, natural killer cells (NK) have recently gained attention as an important type of innate immune regulatory cell. NK cell immunotherapy approaches have been translated into clinical applications, and clinical trials of NK cell infusion in patients with hematological malignancies (HM) and solid tumors have thus far yielded many encouraging clinical results. Understanding the pattern of NK expression and the relationship to different states of NK cells may have direct relevance for immune responses in cancer. Approaches capable of simultaneously detecting NK cells with detailed information on NK differentiation state, however, remain limited. In this study, we used MultiOmyx hyperplexed immunofluorescence (IF) assay to classify and characterize the spatial arrangement of NK cell markers in a pan-cancer cohort including 9 tissue microarrays (TMAs) from breast, head and neck, prostrate, colon, bladder, lung, kidney, ovarian and melanoma indications. The panel includes CD3, CD4, CD8, CD16, CD45, CD56, CD57, CD137, FoxP3, Granzyme B, HLA-E, NKG2A, NKP46 and tumor segmentation marker. The panel enables the detection of NK cells expressing CD56 and/or NKP46 in the 9 different tumor indications. We also studied the expression of activating and inhibiting receptors, such as CD16 and NKG2A, in NK cell population. The active cytotoxic subsets of mature NK cells were evaluated using co-expression of NK cell surface markers with CD57 and the cytotoxic molecule expression in NK cells was assessed using co-expression with Granzyme B. Using proprietary deep-learning-based image analysis, we were able to quantify the densities of these different NK cell population and study the prevalence of these NK cells in different cancer indications included in this study. Many strategies have been developed for exploiting NK-mediated anti-tumor activities. CAR-NK cell therapy and antibodies that directly target NK cell inhibitory receptors such as NKG2A and TIGIT, are currently being evaluated in the clinical trials. The MultiOmyx NK cell panel reported in this study enables the comprehensive profiling of the NK population and can provide greater understanding of NK cell biology during cancer progression. The panel can be further used to explore the efficacy of the NK cell-based immune therapy. Citation Format: Erinn A. Parnell, Jiong Fei, Harry Nunns, Eric Leones, Elaine Yeung, Blair Russell, Flora Sahafi, Qingyan Au. Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4688.
Abstract 2707: Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model
Authors: Martin Ramos-Gonzalez, Eduardo Vázquez-Garza, Gerardo García-Rivas, Cristian Rodríguez-Aguayo, Arturo Chávez-Reyes
Journal: Cancer Research (2023)

Abstract Introduction: Cancer is considered among the main causes of death worldwide and the number of cases keeps rising every year, as there is not an effective treatment to offer for most of them. The lungs represent a frequent target of primary and metastatic tumors due to his high oxygenation and large air spaces to grow. Melanoma is the most aggressive type of skin cancer showing a high affinity to metastasize into the lungs, being the overexpression of the WT1 protein associated with the development of this cancer, as well as several other types. Methods: In this study, we evaluated the effects of siRNA silencing the WT1 protein in both, in vitro in the B16F10 melanoma cell line, and in vivo in a murine model of pulmonary metastatic melanoma implementing a novel delivery strategy of neutral DOPC liposomal-siRNA system administrated via inhalation. Results: In vitro studies showed an effective silencing of WT1 protein (86%) in the siRNAsWT1 treated cells when compared with controls, resulting in a loss of viability (40%) and proliferation (49%) of the cells, induced G1 cell-cycle arrest (11%), a restriction of migration (87%) and invasion (100%) capacities, as well as an activation of apoptosis (12%). In vivo, we tested our delivery system obtaining an efficient biodistribution in lungs, kidney, liver and spleen. The respiratory administration of LWT1siRNA after two weeks of treatment resulted in silencing of WT1 protein (59%), achieving an important antitumor activity with a 44% reduction in tumor weight. In the survival study 80% of the L-WT1siRNA treated mice were alive after all the controls mice had died, delaying the death almost a week at the end of the study. Conclusions: This work demonstrates the efficacy of the neutral DOPC liposomal-siRNA delivery system by respiratory administration as a novel therapy to reduce the malignancy of pulmonary tumors and to increase survivability by silencing specific cancer biomarkers as WT1. Citation Format: Martin Ramos-Gonzalez, Eduardo Vázquez-Garza, Gerardo García-Rivas, Cristian Rodríguez-Aguayo, Arturo Chávez-Reyes. Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2707.
Abstract 2384: Quantitative phase imaging provides new insights into diverse morphologies in cell lines and their behavior in response to alternative cancer treatments
Authors: Rida Khan, Meaghan McDonald, Albina Mikhaylova, Fatima K. Rehman
Journal: Cancer Research (2023)

Abstract Quantitative phase imaging (QPI) through holographic time-lapse microscopy allows for non-invasive measurements of cell dry mass and the ability to track cells for long periods of time with minimal damage, making it most beneficial for research on cancer treatment. Research with this enhanced method of observation can help decipher various morphologies present in specific cell lines and show how each cell can respond differently to provided signals. In this assay, we studied N2a murine neuroblast differentiation in response to Retinoic acid, as well as the anticancerous activity of Saffron and UV in MDA-MB-468 breast carcinoma and HCT116 colon carcinoma cell lines. Cells were observed using Telight’s Q-Phase, a multimodal microscope that utilizes QPI holographic time-lapse technology with label-free monitoring. The cells were able to survive in optimum conditions for up to 96 hours without any appreciative loss of viability. With the emergence of phytochemicals being used as alternative cancer treatments, specific pigments in saffron have shown therapeutic effects against cancer tissues such as apoptosis and decreased carcinogen presence. A microanalysis of the effects of saffron on reducing the invasion potential, viability, and tumorigenicity of breast cancer cell lines was performed. QPI microscopy was performed on MDA-MB-468 human breast adenocarcinoma and HCT116 colon carcinoma cell lines treated with varying doses of saffron and ultraviolet radiation. Cell lines were observed under the microscope for 58-hour periods for changes in mass, movement, and replication. The quantitative data showed a reduction of projections from saffron treatment but did not show any significant reduction in cell movement over the 58-hours. These results were later confirmed through scratch wound and Boyden chamber assays. Similarly, when examining the process of N2a neuroblast differentiation, it was observed that cells do not move towards differentiation in a linear manner, but rather changed their direction and path several times before committing to their final phenotype. These types of observations are critical in understanding the biological processes behind differentiation and drug response and could not have been done without using the various quantitative and analysis tools provided by the QPI technology. Citation Format: Rida Khan, Meaghan McDonald, Albina Mikhaylova, Fatima K. Rehman. Quantitative phase imaging provides new insights into diverse morphologies in cell lines and their behavior in response to alternative cancer treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2384.
Abstract 2538: MiR-92b targets FBXW7 in GBM cells
Authors: Annelis Odette Sanchez-Alvarez, Nilmary Grafals-Ruiz, Yasmarie Santana-Rivera, Fatma Valiyeva, Nathaly M. Rosado-Rivera, Pablo E. Vivas-Mejía
Journal: Cancer Research (2023)

Abstract MicroRNA (miRNA) dysregulation plays a central role in the initiation, progression, and drug resistance of virtually all cancer types. MiRNA-based therapies are a promising therapeutic modality against most malignancies. MiR-92b is aberrantly abundant in glioblastoma (GBM), the most aggressive and mortal of all primary brain tumors. Thus, miR-92b is a potential target for GBM therapy. First, it is critically important to assess the molecular mechanism of miR-92b regulation in GBM cells. In this study, we investigated the molecular and biological effects of targeting miR-92b in GBM cells with oligonucleotide miRNA inhibitors (OMIs). Incubation of GBM cells with miR-92b-OMIs resulted in decreased cell proliferation and reduction in cell migration. MiR-92b inhibition also promoted apoptosis as assessed by caspase-3 activation. Bioinformatics and real-time PCR studies identified several miR-92b target genes in GBM cells. Western blot analysis and luciferase reporter assays confirmed F-Box and WD Repeat Domain Containing 7 (FBXW7) as a direct miR-92b target gene in GBM cells. FBXW7 is one of ~70 F-BOX proteins in humans particularly studied in cancer due to its role in the ubiquitylation of positive regulators; also known as accelerators of the cell cycle (i.e. the oncogene c-Myc). Thus, an increase in FBXW7 promoted the decrease of c-MYC protein levels in GBM cells. These findings indicate that targeting miR-92b inhibits GBM cell proliferation by increasing the levels of tumor suppressor genes in charge of protein ubiquitination. Citation Format: Annelis Odette Sanchez-Alvarez, Nilmary Grafals-Ruiz, Yasmarie Santana-Rivera, Fatma Valiyeva, Nathaly M. Rosado-Rivera, Pablo E. Vivas-Mejía. MiR-92b targets FBXW7 in GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2538.
Abstract 1834: Cholinergic nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs
Authors: Laurel B. Darragh, Tiffany T. Pham, Michael M. Knitz, Alexander Nguyen, Laith A. Mukdad, Yazeed Alhiyari, Maie St. John, Sana D. Karam
Journal: Cancer Research (2023)

Abstract Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). We investigate how nerves within the tumor microenvironment impact the adaptive immune system and tumor growth. RNA sequencing and proteomics data led us to identify cholinergic nerves as the potential drivers of tumor growth in human and mouse HNSCC. Using three syngeneic orthotopic murine models of HPV-unrelated HNSCC, experimental and pharmacologic manipulation of cholinergic output, and knockout mouse models, we discovered that activation of cholinergic nerves accelerate tumor growth by directly acting on the adaptive immune system. Cholinergic nerve release of calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking cholinergic nerves pharmacologically or genetically increased CD8 T cell activity in vivo. These data support therapeutic translation of cholinergic nerve targeting of CGRP release to enhance antitumor immune response within the HNSCC TME. Citation Format: Laurel B. Darragh, Tiffany T. Pham, Michael M. Knitz, Alexander Nguyen, Laith A. Mukdad, Yazeed Alhiyari, Maie St. John, Sana D. Karam. Cholinergic nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1834.